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The purpose of the present study was to evaluate the effects of season on the in vitro fertilizing ability of bovine spermatozoa and subsequent embryo development. Bovine oocytes were matured and fertilized in vitro with Holstein dairy bull sperm cells collected and frozen in different seasons (winter, spring, and summer). On d 2 and 8 postinsemination, cleavage and blastocyst rates, respectively, were recorded; the blastocysts were graded for morphology. The number of sperm cells binding to the zona pellucida of oocytes, together with the number of nuclei in the developing blastocysts, were assessed after staining with Hoechst. No significant differences were observed among seasons in cleavage and embryo development rate. However, the proportion of "advanced blastocysts" was significantly higher in spring compared with winter and summer, with a corresponding decrease in the proportion of early blastocysts in spring compared with winter and summer. The number of sperm cells binding per oocyte was significantly lower in the oocytes inseminated with sperm samples collected in summer compared with winter or spring. Moreover, a significant interaction was observed in the number of sperm cells binding per oocyte between bull and season. Although no significant differences were observed among seasons in the number of nuclei per blastocyst, a significant interaction was observed between bull and season for this variable. Embryo development rate in in vitro fertilization appeared to be affected by season of semen collection, with sperm samples collected in spring being associated with a higher proportion of advanced blastocysts and better morphology than those collected at other times of the year.

The aim of the present study was to make a retrospective analysis of the relationship between climatic factors and sperm quality of frozen-thawed semen from bulls kept in temperate climates. Semen samples from 21 European dairy bulls from 2 countries were collected and cryopreserved in winter, spring, and summer. Sperm quality parameters such as kinematics, morphology, plasma membrane integrity, mitochondrial membrane potential, sperm chromatin structure assay, and reactive oxygen species were analyzed and correlated retrospectively with climate factors recorded by the local meteorological office. This study demonstrated that sperm quality parameters are more likely to be correlated with climate factors 1 or 2 mo before semen collection than in the month of semen collection. During the month of sperm collection, sperm kinematics, DNA fragmentation, and hydrogen peroxide production were the only sperm quality parameters related to climate factors, whereas 1 and 2 mo before sperm collection, normal morphology and additional sperm kinematics, in addition to DNA fragmentation and hydrogen peroxide production, were correlated with climate factors. In conclusion, dairy bull sperm quality is affected by climatic conditions, even in so-called temperate zones. The timing of heat stress during spermatogenesis determines which aspects of sperm quality are likely to be affected. Husbandry conditions for bulls used for semen collection should be adapted to allow the animals' physiological responses for temperature regulation within the scrotum to operate fully, to mitigate the effects of increased temperature and humidity. Extremes of temperature should be avoided.

Simple Summary The monitoring of a horse’s endocrine status can provide valuable information of its welfare and sexual and health conditions. The use of non-invasive matrixes for hormonal monitoring, as alternatives to blood samples, are being increasingly used. Among them, the measurement of hormones deposited in the hair shaft has been widely used as a retrospective hormonal biomarker. In the present study, we aimed to evaluate the seasonal variations in hair C, T and DHEA-S in horses for a whole year, as well as to assess the variations between seasons of C/DHEA-S and T/C ratios as a retrospective measure of the hypothalamic–pituitary–adrenal and hypothalamic–pituitary–gonadal axis activity. The results showed how cortisol, testosterone and dehydroepiandrosterone sulphate and their ratios were significantly affected by season. Therefore, as shown in this study, season should be considered when analysing sexual and stress hormones in stallion hair. Abstract The monitoring of stress physiology includes studying a wide range of endocrinological mechanisms, which can be assessed using multiple tissue samples. This study aimed to evaluate the seasonal variations of hair C, T and DHEA-S in horses for a whole year, as well as to assess the variations between seasons of C/DHEA-S and T/C ratios as a retrospective measure of the hypothalamic–pituitary–adrenal and hypothalamic–pituitary–gonadal axis activity. Ten pure-breed Menorca stallions were included in the study. The hair samples were collected approximately every two months following the shave-reshave method caudally to the sternum. After a methanol-based extraction, samples were analyzed by enzyme immunoassay for cortisol, testosterone, and dehydroepiandrosterone sulphate. Following our findings, we detected that cortisol, testosterone and dehydroepiandrosterone sulphate were significantly affected by seasonality, with the highest values of cortisol during summer and the lowest values of testosterone during spring. Dehydroepiandrosterone sulphate concentrations were increased in autumn compared to the other studied periods. Additionally, the studied hormone ratios showed variations between seasons. To conclude, season should, therefore, be considered when assessing sexual and stress hormones in stallion hair, since this variable can be a potential influencing factor and led to misinterpretations.

Monitoring the hypothalamic–pituitary–adrenal (HPA) axis through determination of fecal cortisol metabolite (FCM) levels is a non-invasive method useful for understanding how handling and social conditions may affect the physiological status of zoo animals. The present study used FCM analysis to evaluate whether the HPA axis activity of a lion pride was modified by a change in social and handling conditions after the death of the dominant male. Five African lions (Panthera leo bleyenberghi), two males and three females, were included in the study. Fecal samples were collected before and after the death of the dominant male. To avoid cohabitation conflicts between males before the dominant male died, subgroups were established and subjected to weekly changes between indoor and outdoor facilities. After the death of the dominant male, these management dynamics ceased, and the remaining four lions were kept together outdoors. Significant lower group FCM concentrations (p &lt, 0.001) were detected after the decease of the dominant male, probably associated with a decrease in daily handling, together with a more stable social environment. Overall, the present study indicates the effect of different management scenarios on the HPA axis activity and differentiated physiological responses to the same situation between individuals.

Simple Summary Multi-unowned-cat environments can be highly stressful for cats, and infectious and parasite diseases spread quickly and are difficult to prevent. This study aimed to determine the occurrence of intestinal parasites and fecal cortisol metabolites (FCM) in cat feces collected from different multi-cat environments and assess the effect of housing conditions on intestinal parasites and FCM levels in order to develop more efficient control strategies. Cat fecal samples from rescue shelters, catteries and feline colonies were analyzed with coprological methods to detect intestinal parasite patency and determine FCM. Helminth infection was mainly detected in free-roaming cats. In confined cats, protozoa infections were more likely detected in shelter cats than in cattery cats. Exposure to dogs was associated with parasite infection and cats highly exposed to dogs with visual contact and audible barking showed higher intestinal protozoa prevalence than cats that were not exposed to dogs. FCM levels were correlated with enclosure size and protozoa infection. Reducing stress by improving housing in terms of enclosure size and avoiding exposure to dogs may have an impact on the occurrence of intestinal parasites, especially protozoa. Abstract Housing conditions were assessed in different unowned multi-cat management models in order to evaluate their impact on the occurrence of intestinal parasites and fecal cortisol metabolite (FCM) levels. Fresh stool fecal samples were collected from rescue shelters, catteries and feline colonies for coprological analyses in order to detect intestinal parasite patency and fecal cortisol metabolites. A questionnaire provided information about the facilities, management and housing conditions of cats, including information about dog exposure, enclosure size, environment enrichment and changes to group composition. Overall, intestinal parasite infection was detected in 58.2% of fecal samples collected. The occurrence of intestinal parasites detected in free-roaming cats was 82.2%, mainly due to helminth infection. The parasite infection rate was 57.3% in rescue shelters and 34.6% in catteries. In confined cats, protozoa infection was more likely detected in rescue shelters than in catteries (RR = 2.02 (1.30–3.14), p = 0.0012). Although the FCM values were very variable between cats, the enclosure size and parasite infection were correlated with the average FCM. A small enclosure size was correlated with high fecal cortisol metabolites (p = 0.016). Protozoa-positive samples showed higher FCM levels than negative samples (p = 0.0150). High dog exposure was statistically associated with protozoa infection (p = 0.0006). The results indicated that improving housing, especially in terms of floor space and avoiding dog exposure, reduces stress and can thus be applied to make control strategies in multi-unowned-cat environments more efficient, especially when cats are confined.

Presence of the hyaluronan (hyaluronic acid, HA) receptor CD44 on spermatozoa has been difficult to pursue, mostly obeying to the use of different commercial mono- and/or polyclonal antibodies, often lacking proper controls. Here, we describe how the presence (Western blotting) and specific location (immunocytochemistry) of the CD44 receptor differs in ejaculated pig spermatozoa depending on the type of antibody and protocol used. While we were able to detect binding to spermatozoa and mark its presence in the sperm membrane, the use of blocking peptides clearly indicated that only the monoclonal antibody could confirm the specific presence and location of the CD44 receptor, whereas the polyclonal antibody was detecting multiple presumed CD44 isoforms or degraded proteins thus proving unspecific. These results call for strict protocols when attempting immunological determination of sperm membrane receptors. Funding Agencies|Lions Forskningsfond 2016-postdoc, Linkoping, Sweden [DNR LIU-2016-00641]; Forskningsradet i Sydostra Sverige [473121, 745971]; Svenska Forskningsradet Formas [2017-00946]

letrozole is an aromatase inhibitor that stops the production of estrogen through interrupting the entrance of hormone androgen into a small amount of estrogen. Therefore, the current study was developed to estimate orally administrated Letrozole on the reproductive performance and relative abundance of Foxj1, PVRL3, and LPR2 mRNA in aged roosters. Fifty-five-week old ROSS 308 breeder roosters (n = 18) were orally treated using letrozole. Primarily, the body weight of the animals was recorded, and they were randomly classified into three groups (n = 6 birds/group) receiving different doses of Letrozole, including 0, 0.015, and 0.03 mg/kg body weight/day for three weeks. At the end of the trial, seminal traits, plasma, testicular hormone levels (testosterone, estradiol, and FSH), histopathological studies, in vitro fertility, and relative abundance of testis PVRL3, epidydimal Foxj1, and LPR2 mRNA were evaluated. Based on the results, the sperm quality variables were statistically higher in the 0.03 group compared to the controls. Greater histologic parameters, such as diameter of seminiferous tubules, thickness of seminiferous epithelium, categorized epididymal region, and in vitro fertility rates were estimated for the treated groups(p 0.001). Plasma and testicular testosterone, estradiol concentrations, and plasma FSH levels were significantly influenced by letrozll treatment (p 0.001). Relative mRNA transcript abundance increased for PVRL3 and decreased for Foxj1 and LPR2 in treated groups. Overall, aromatase inhibitors can enhance the reproductive performance of aged commercial broiler breeder roosters. However, it can impact endocytosis and ciliogenesis events via reducing estradiol.

Contents Heat-shock protein 70 (HSP70) plays a crucial role as intracellular cytoprotectant and molecular chaperone. A phenomenon of heat stress (HS) leads to production of these proteins that could be beneficial to cells during cryopreservation, which is also a stressful process for the cell. This study aimed to evaluate the protective effect of exposure of bovine oocytes to moderate HS during in vitro maturation (IVM) prior vitrification. First, oocytes were subjected to HS (41.5°C for 1 hr) at 0, 4, 8, 12 or 16 of IVM. Oocytes in vitro matured for 20 hr served as control group. Presence of HSP70 was detected at 20 hr by immunofluorescence. HSP70 expression was significantly higher when oocytes were subjected to HS at 8 hr of IVM. Next, oocytes were distributed into four groups: Control: IVM oocytes; VIT: oocytes vitrified/warmed at 20 hr of IVM; HS: oocytes subjected to HS at 8 hr of IVM; HS-VIT: oocytes subjected to HS at 8 hr of IVM and vitrified/warmed at 20 hr of IVM. Oocytes were fertilized at 24 hr of IVM, and cleavage and blastocyst yield were assessed. No significant differences were observed among treatments when cleavage rate was evaluated. However, fresh control and HS oocytes resulted in a significantly higher (p

Simple Summary Greater Flamingos are commonly kept under flight restraint in zoos. Some are pinioned, others only feather clipped and some remain physically intact but live in aviaries that are often not large enough to fly. In this study, we compared these three groups by measuring corticosterone (a hormone associated with stress) in their feathers in order to find out which of the restraining methods is most compatible with animal welfare. Additionally, we carried out behavioral observations on all groups to detect potential stressors other than the status of flight itself. We expected to find differences in CORTf between deflighted and airworthy flamingos. However, no significant differences in feather corticosterone were measured between the three groups and the hypothesis was rejected. The most important factor for the level of corticosterone was found to be the zoological institution itself, reflecting the housing conditions. We hypothesize that the method by which a Greater Flamingo is hindered from flying does not have measurable effect on the corticosterone concentration in its feathers. Although these findings suggest that all methods are equally impacting animal welfare, we highlight the need for further improved studies based on this model. Abstract Deflighting zoo birds is a practice that receives increasing criticism due to its presumed incompatibility with animal welfare. To our knowledge, this is the first approach to address this problem in a scientific way. To do this, we compared feather corticosterone (CORTf) from Greater Flamingos (Phoenicopterus roseus, n = 151) of different flight status (i.e., pinioned, feather clipped, airworthy) from twelve different zoological institutions. To complement the hormone measurements, behavioral observations (scan samplings) were conducted prior to feather sampling within the presumed time frame of feather growth. We hypothesized that CORTf of the deflighted flamingos would differ from CORTf of their airworthy conspecifics. No significant difference in CORTf was found between the three groups, and our hypothesis was rejected. However, the impact of the institution itself (i.e., the housing conditions) proved to be the most dominant variable (variance between the institutions = 53.82%). Due to high variability, the behavioral observations were evaluated descriptively but did not give rise to doubt the findings in CORTf. Therefore, we assume that the method of flight restraint of Greater Flamingos does not have a measurable effect on CORTf. We consider this model for evaluating animal welfare of zoo birds a useful tool and provide ideas for further adjustments for consecutive studies.

Horse transportation for temporary relocation during rest periods is a common and widespread practice among horse owners, either from sport competition or working tasks. This study aimed to determine the effect of a relocation period and the multiple factors associated with a rest period on hair cortisol concentrations (HCCs) in horses. Additionally, this study reports the seasonal effect on HCCs and hair growth over a year. Thirteen police horses, Pure Spanish stallions of various ages (5&ndash, 13 y), were selected to participate in this study. Hair sample collection was carried out approximately every 30 d for seven months (Study 1) and a year (Study 2). Cortisol determinations were performed by enzyme immunoassay. Interestingly, Study 1 revealed that relocated horses (n = 4) exhibited elevated HCCs compared with control horses (n = 4) after the relocation period (p &lt, 0.05). Study 2 (n = 5) showed higher HCCs during summer compared with autumn and winter, and higher hair growth rates in winter compared with the other seasons (p &lt, 0.05). Relocated horses had higher HCCs, suggesting a change in their welfare status, probably related to the sudden change in their surrounding conditions. However, these results should be interpreted cautiously due to the low sample size used. The nature of the relationship between HCCs and horse welfare needs to be further examined.

The avian seminal fluid (SF) is a protein-rich fluid, derived from the testis, the rudimentary epididymis and, finally, from the cloacal gland. The SF interacts with spermatozoa and the inner cell lining of the female genital tract, to modulate sperm functions and female immune responsiveness. Its complex proteome might either be free or linked to extracellular vesicles (EVs) as it is the case in mammals, where EVs depict the tetraspanin CD9; and where those EVs derived from the epididymis (epididymosomes) also present the receptor CD44. In the present study, sperm-free SF from Red Jungle Fowl, White Leghorn and an advanced intercross (AIL, 12th generation) were studied using flow cytometry of the membrane marker tetraspanin CD9, Western blotting of the membrane receptor CD44 and electron microscopy in non-enriched (whole SF) or enriched fractions obtained by precipitation using a commercial kit (Total Exosome Precipitation Solution). Neither CD9- nor CD44 could be detected, and the ultrastructure confirmed the relative absence of EVs, raising the possibility that avian SF interacts differently with the female genitalia as compared to the seminal plasma of mammals.

Simple Summary The analysis of circulating cortisol has been by far the most common method used as a means to assess fish stress responses and, thus, animal welfare. To avoid many of the drawbacks inherent to blood sampling, cortisol can be less-invasively detected in fish skin mucus. The measurement of cortisol in skin mucus however, has, to date, only been demonstrated as suitable for farm fish, although its application to free-ranging animals would offer many advantages. The present study was therefore designed to evaluate the applicability of skin mucus cortisol analysis as a potential tool to assess habitat quality. To that end, wild fish residing in environments of different habitat quality were sampled for blood and skin mucus. First, several physiological endpoints typically used as indicators of exposure to pollutants were accurately related to the habitat quality in the Catalan chub (Squalius laietanus). Second, cortisol levels in blood were also compared between habitats, and they were successfully correlated to skin mucus cortisol concentrations. Finally, we contrasted the patterns of response of all the endpoints assessed to skin mucus cortisol levels across the sites. The strong linkages detected in this study provide new evidence that the measurement of cortisol in skin mucus could be potentially used as a biomarker of habitat quality in freshwater fish. Abstract Cortisol levels in fish skin mucus have shown to be good stress indicators in farm fish exposed to different stressors. Its applicability in free-ranging animals subject to long-term environmental stressors though remains to be explored. The present study was therefore designed to examine whether skin mucus cortisol levels from a wild freshwater fish (Catalan chub, Squalius laietanus) are affected by the habitat quality. Several well-established hematological parameters and cortisol concentrations were measured in blood and compared to variations in skin mucus cortisol values across three habitats with different pollution gradient. Fluctuations of cortisol in skin mucus varied across the streams of differing habitat quality, following a similar pattern of response to that detected by the assessment of cortisol levels in blood and the hematological parameters. Furthermore, there was a close relationship between cortisol concentrations in skin mucus and several of the erythrocytic alterations and the relative proportion of neutrophils to lymphocytes. Taken together, results of this study provide the first evidence that skin mucus cortisol levels could be influenced by habitat quality. Although results should be interpreted with caution, because a small sample size was collected in one studied habitat, the measurement of cortisol in skin mucus could be potentially used as a biomarker in freshwater fish.

The aim of this study was to induce the cold-inducible RNA-binding protein (CIRBP) expression on cumulus-oocyte complexes (COCs) through exposure to a sub-lethal cold shock and determine the effects of hypothermic temperatures during the in vitro maturation of bovine oocytes. Nuclear maturation, cortical granule redistribution and identification of cold-inducible RNA-binding protein (CIRBP) were assessed after 24 hr of in vitro maturation of control (38.5 degrees C) and cold-stressed oocytes (33.5 degrees C). The presence of CIRBP was assessed by Western blot in COCs or denuded oocytes and their respective cumulus cells. Based on the odds ratio, cold-stressed oocytes presented higher abnormal cytoplasmic distribution of cortical granules and nuclear maturation than the control group. Although CIRBP was detected in both control and cold-stressed groups, cold-stressed COCs had 2.17 times more expression of CIRBP than control COCs. However, when denuded oocytes and cumulus cells were assessed separately, CIRBP only was detected in cumulus cells in both groups. In conclusion, cold shock induced CIRBP expression, but it negatively affected nuclear maturation and cortical granule distribution of bovine oocytes. Moreover, the expression of CIRBP was only identified in cumulus cells but not in oocytes. Funding Agencies|FI grant [2018 FI_B 00236]; [AGL2016-79802-P]; [AGL2016-81890-REDT]; [FPU2015/06029]; [IJCI-2015-24380]

In recent times, the detection of cortisol in hair is intended to be used as an animal-based indicator for the assessment of chronic stress. However, the relationship between the ability to report average values of long-term circulating cortisol concentrations and the sensitivity to acute peaks of cortisol is still unclear. To gain insight into this relationship, 24 Holstein-Friesian bull calves under the same management conditions were used in this study. Two injections of ACTH (at D0 and D7) were administrated to twelve animals with the aim to create two acute increases of serum cortisol concentrations. Blood samples were taken in order to determine the duration of serum cortisol peaks and to confirm a mediated response by the administration of ACTH. Cortisol concentrations from 14-day-old white hair samples collected from forehead and hip were analysed separately by EIA and compared with those from the control group. Serum cortisol analyses revealed an acute increase of cortisol concentrations for approximately three hours after each ACTH administration. Concentrations of hair cortisol from forehead and hip locations showed no differences between ACTH-administrated and control animals. Hair cortisol concentrations from 14-day old samples were not altered by two acute elevations of serum cortisol suggesting that hair cortisol is not masked by short and non-recurrent moments of stress. These results are a step forward in the validation of hair cortisol detection as a robust integrative measure of serum cortisol concentrations from an extended period of time.

In the warm months the function of the spermatozoa can be affected by the temperature of the reproductive tract of the female exposed to hyperthermic conditions. The aim of this study was to evaluate the impact of heat stress on sperm parameters in an in vitro model and to determine if there were seasonal effects on sperm heat tolerance. Sperm samples from 32 New Zealand White rabbits were collected in two seasons and incubated at scrotal (32.5°C), body (37°C) or hyperthermic (42°C) temperatures for 3h. Sperm viability and morphology were evaluated using nigrosin-eosin staining. Motility and metabolic activity parameters were determined using computer-assisted sperm analysis and the QBlue cell viability test, respectively. The incubation of spermatozoa at 42°C decreased (P0.05) the mean values of total motility, curvilinear (VCL) and mean velocity (VAP) as well as the metabolic activity with respect to the incubation at 32.5°C and 37°C. No seasonal effects were observed except for the highest percentages of bent and coiled tails in the cold season, and the highest mean values of VCL, linear velocity and VAP in the warm season (P0.01). The interaction between in vitro heat stress and season was significant for metabolic activity (P=0.02). Our results suggest that rabbit spermatozoa parameters are largely modified by a short exposure to hyperthermic conditions, in terms of metabolic activity and motility parameters. Thus, a short exposure of spermatozoa to an environment of 42°C in temperature for only 3h may compromise sperm functionality. Additionally, sperm metabolic activity is influenced by season.

Ensuring welfare in captive wild animal populations is important not only for ethical and legal reasons, but also to maintain healthy individuals and populations. An increased level of social behaviors such as aggression can reduce welfare by causing physical damage and chronic stress to animals. Recently, cortisol in hair has been advanced as a non-invasive indicator to quantify long-lasting stress in many species. The sensitivity of social behavior and hair cortisol concentration was evaluated in several groups of dorcas gazelles (Gazella dorcas). Four different groups of gazelles from three different zoos were observed and the expression of intra-specific affiliative and negative social behaviors was assessed across the different groups. Hair samples were taken from sub-groups of animals and analyzed for cortisol concentrations. Significant differences between groups of dorcas gazelles were found in frequency of negative social behavior and hair cortisol concentration. Despite the low sample size, these two parameters had a positive Spearman correlation coefficient (rs = +0.80, P = 0.20). These results suggest that hair cortisol levels are sensitive to differences in the social structure of dorcas gazelles. Zoo Biol. 35:467–473, 2016. © 2016 Wiley Periodicals, Inc.

The Dorcas gazelle (Gazella dorcas), in the family Bovidae, is one of the smallest species of the genus Gazella. Its population is decreasing and it is classified as vulnerable by the International Union of Conservation of Nature Red List of Threatened Species. The subspecies Saharan Dorcas gazelle (G. dorcas osiris), originating in the central-western Saharan and Sahel regions, is almost extinct in the wild because of over-hunting. Thereon, assisted reproductive techniques (ARTs) could help in its population management. Here, we performed invitro maturation (IVM) of oocytes recovered from an adult female of Saharan Dorcas gazelle (10 years old) to attempt IVF with frozen-thawed epididymal sperm cells from an adult male (9 years old) of the same subspecies, following protocols for ARTs in cattle. Epididymal sperm was obtained after postmortem examination by cannulation of vas deferens with commercial semen extender Gent A (Minitub). Motility features were assessed using a computer-assisted sperm analysis system (Proiser SL), and viability was evaluated using eosin/nigrosin staining. After collection, viability (88.3%), total (17.8%) and progressive (8.7%) motility were assessed. A total of 820.5×106 sperm cells was obtained and diluted (1:1) in a semen extender with permeable cryoprotectants (Gent B, Minitub). Then, the sample was refrigerated in straws (200×106 mL−1; 0.5mL) at 4°C for 1h and exposed to liquid nitrogen vapors (LN2) for 10min before being directly plunged into and kept on LN2. A total of 35 oocytes were recovered by slicing, 27 of which were selected for IVM (24h, 38.5°C, 5% CO2 in humidified air). Motile sperm were obtained by centrifugation of thawed semen (10min, 1000×g) by performing a density gradient (BoviPure, Nidacon). Post-thawing viability (72.9%) and total (14%) and progressive (4%) motility were evaluated just before IVF. After IVM, 17 oocytes were incubated for IVF for 20h with 1×106 thawed sperm cells mL−1 (although the first polar body extrusion was observed only in 5 of the 17 oocytes before IVF; 29.4%). Also, 7 oocytes selected for IVM were vitrified and kept for the genetic bank, and 3 were fixed. Thereafter, all oocytes that underwent IVF were incubated for invitro embryo culture (38.5°C, 5% CO2, 5% O2 in humidified air) to avoid discarding any potential zygote. Cleavage was assessed at 48h post-IVF. From those, only 2 oocytes successfully achieved IVF out of the 5 that showed first polar body extrusion (40%), although cleavage was asymmetric and embryos did not progress. The rest of oocytes (12) did not achieved IVM, IVF, or cleavage. Results suggest that optimal protocols and appropriate individualized conditions for vulnerable nondomestic ungulates are required for successful ARTs. Future efforts are needed to improve preservation strategies for the gametes/embryo conservation of endangered species. JG and MRC contributed equally to this work. JG is supported by AGAUR (2018 FI_B 00236) and MRC by FPU (Training for Academic Staff) (FPU15/06029).

Rooster fertility peaks between 30 and 40 weeks of age and declines rapidly from 50 weeks of age. This is linked to a reduction in the number of spermatozoa in the ejaculate due to high density of spermatids in the seminiferous tubules of low-fertility roosters. In this study, we assessed the effects over spermatogenesis of both letrozole, an aromatase inhibitor, and a commercial herbal extract designed for improving fertility composed by Tribulus, Cinnamomum, Zingiber and Sativus. Forty-two-week-old Ross 308 roosters (n = 24) were distributed into four groups: control, letrozole (0.03 mg/kg), herbal extract (0.04 ml/kg) and letrozole+herbal extract. After 14 weeks of daily oral supplementation, their testes and epididymides were weighed, fixed and sectioned for assessment of spermatogenesis and quantification of sperm cells inside the lumen. Differences in seminiferous tubules measurements and density of sperm cells were tested using R software (version 3.0.1). Although body weight was not affected by the treatment, testes from animals treated with the combination of letrozole and herbal extracts were heavier than those from control animals. Animals treated with either letrozole or herbal extract, or their combination, showed a significant higher number of sperm cells inside the seminiferous tubules and epididymis than control animals (p

Contents Oocyte vitrification causes less cell stress than slow cooling, but cytoskeletal and spindle alterations may occur affecting the oocyte competence. In vitro maturation (IVM) supplementation with different antioxidant molecules has been performed to attenuate this harmful stress. Coenzyme Q10 (CoQ10) supplementation has previously shown to have positive effects in bovine and mouse in vitro embryo development. The aim of this study was to evaluate the effects of CoQ10 during bovine oocyte IVM and vitrification. Cumulus–oocyte complexes (COCs) (n = 311) were cultured under standard maturation conditions with 0 μM (control), 25 μM and 50 μM CoQ10 supplementation. After 22 hr, a cohort of 170 oocytes both from the control and from CoQ10-supplemented groups were vitrified, warmed and returned to incubation until 24 hr of maturation, while the rest of the oocytes (n = 141) remained fresh. Then, oocyte survival was assessed morphologically by stereomicroscopy. Oocytes from all groups were then fixed and stained for assessing cortical granules (CG) migration and nuclear stage. High rates of oocyte MII progression and appropriate CG migration as a continuous layer beneath the plasma membrane were obtained both in control and in CoQ10 groups. Results showed that although vitrification has great impact in survival of IVM bovine oocytes, 50 μM CoQ10 supplementation significantly improved oocyte survival (p = .045) and reduced the premature CG exocytosis, helping to preserve the CG migration pattern (31.3% control vs. 54.5% in 50 μM CoQ10; p = .039), attenuating the negative effects of vitrification.

7 pages, 2 figures, 3 tables, supplementary data https://doi.org/10.1016/j.ygcen.2019.01.015, A significant body of literature suggests that aquatic pollutants can interfere with the physiological function of the fish hypothalamic-pituitary-interrenal (HPI) axis, and eventually impair the ability to cope with subsequent stressors. For this reason, development of accurate techniques to assess fish stress responses have become of growing interest. Fish scales have been recently recognized as a biomaterial that accumulates cortisol, hence it can be potentially used to assess chronic stress in laboratory conditions. We, therefore, aimed to evaluate the applicability of this novel method for cortisol assessment in fish within their natural environment. Catalan chub (Squalius laietanus) were sampled from two sites; a highly polluted and a less polluted (reference) site, in order to examine if habitat quality could potentially influence the cortisol deposition in scales. We also evaluated the seasonal variation in scale cortisol levels by sampling fish at three different time points during spring-summer 2014. In each sampling, blood was collected to complement the information provided by the scales. Our results demonstrated that blood and scale cortisol levels from individuals inhabiting the reference site were significantly correlated, therefore increasing the applicability of the method as a sensitive-individual measure of fish HPI axis activity, at least in non-polluted habitats. Since different environmental conditions could potentially alter the usefulness of the technique, results highlight that further validation is required to better interpret hormone fluctuations in fish scales. Scale cortisol concentrations were unaffected by habitat quality although fish from the polluted environment presented lower circulating cortisol levels. We detected a seasonal increase in scale cortisol values concurring with an energetically costly period for the species, supporting the idea that the analysis of cortisol in scales reveals changes in the HPI axis activity. Taken together, the present study suggests that cortisol levels in scales are more likely to be influenced by mid-term, intense energetically demanding periods rather than by long-term stressors. Measurement of cortisol in fish scales can open the possibility to study novel spatio-temporal contexts of interest, yet further research is required to better understand its biological relevance

Biological molecules isolated from organisms that live under subzero conditions could be used to protect oocytes from cryoinjuries suffered during cryopreservation. This study examined the cryoprotectant role of exopolysaccharides of Pseudomonas sp. ID1 (EPS ID1) in the vitrification of prepubertal and adult cow oocytes. IVM oocytes were vitrified and warmed in media supplemented with 0, 1, 10, 100 or 1000µgmL−1 EPS ID1. After warming, oocytes were fertilised and embryo development, spindle morphology and the expression of several genes in Day 8 blastocysts were assessed. Vitrification led to significantly lower proportion of prepubertal oocytes exhibiting a normal spindle configuration. In fresh control oocytes and most groups of vitrified adult oocytes, similar percentages of oocytes with a normal spindle configuration were observed. Percentages of Day 8 blastocysts were similar for prepubertal oocytes vitrified in the absence or presence of 1 or 10µgmL−1 EPS ID1 and for adult oocytes vitrified in the presence of 10µgmL−1 EPS ID1 compared with non-vitrified oocytes. EPS ID1 supplementation had no effect on solute carrier family 2 member 3 (SLC2A3), ubiquitin-conjugating enzyme E2A (UBE2A) and histone deacetylase 1 (HDAC1) expression in Day 8 blastocysts form adult oocytes. However, supplementation with 10 and 100µgmL−1 EPS ID1 led to increased expression of genes involved in epigenetic modifications (DNA methyltransferase 3 alpha (DNMT3A) and K (lysine) acetyltransferase 2A (KAT2A)) and apoptosis (BCL2 associated X apoptosis regulator (BAX) and BCL2-like 1 (BCL2L1)). The lowest BAX:BCL2L1 ratio was found in the 10µgmL−1 EPS ID1-supplemented group. The results suggest that 10µgmL−1 EPS ID1 added to vitrification and warming media may help protect bovine oocytes against cryodamage.

The measurement of fecal progesterone metabolites (fPM) by enzyme immunoassay analysis is a non-invasive technique that permits gathering reproductive information from wildlife without the stress associated with restraint. In the collared peccary (Pecari tajacu), a high correlation between serum progesterone and fPM levels (r2 = 0.783) suggests that fPM can be used to monitor their reproductive function. We monitored fPM during the estrous cycle of 15 collared peccary females. Estrous cycles averaged 27.9 ± 4.5 days (n = 28), ranging from 21 to 36 days. The luteal phase was 22.2 ± 4.8 days and the inter-luteal phase was 4.3 ± 1.4 days. Mean concentration of fPM across pregnancy were not different from those found during the luteal phase (1230 ± 718 and 1265 ± 584 ng/100 mg dried feces, respectively), however, significant differences were found when luteal phase concentrations were compared only against fPM concentrations during late pregnancy. In addition, late pregnancy fPM concentrations (1893 ± 551 ng/100 mg) were also significantly higher than those in the early (639 ± 339 ng/100 mg) and mid (1134 ± 449 ng/100 mg) pregnancy. For females during the early post-partum period, fPM concentrations were significantly increased (243 ± 118 ng/100 mg) than those of non-cycling females (103 ± 89 ng/100 mg). The analysis of fPM is a simple, non-invasive methodology to detect the ovarian activity in the collared peccary; moreover, it provides a husbandry tool, which may be used to help understand how social structure may impact reproduction.

The zona pellucida (ZP) is an extracellular matrix that surrounds mammalian oocytes. In eutherians it is formed from three or four proteins (ZP1, ZP2, ZP3, ZP4). In the few marsupials that have been studied, however, only three of these have been characterised (ZP2, ZP3, ZP4). Nevertheless, the composition in marsupials may be more complex, since a duplication of the ZP3 gene was recently described in one species. The aim of this work was to elucidate the ZP composition in marsupials and relate it to the evolution of the ZP gene family. For that, an in silico and molecular analysis was undertaken, focusing on two South American species (gray short-tailed opossum and common opossum) and five Australian species (brushtail possum, koala, Bennett’s wallaby, Tammar wallaby and Tasmanian devil). This analysis identified the presence of ZP1 mRNA and mRNA from two or three paralogues of ZP3 in marsupials. Furthermore, evidence for ZP1 and ZP4 pseudogenes in the South American subfamily Didelphinae and for ZP3 pseudogenes in two marsupials is provided. In conclusion, two different composition models are proposed for marsupials: a model with four proteins (ZP1, ZP2 and ZP3 (two copies)) for the South American species and a model with six proteins (ZP1, ZP2, ZP3 (three copies) and ZP4) for the Australasian species.

Hair may be a useful matrix to detect cumulative cortisol concentrations in studies of animal welfare and chronic stress. The aim of this study was to validate a protocol for cortisol detection in hair from dairy cattle by enzyme immunoassay (EIA). Seventeen adult Holstein-Friesian dairy cows were used during the milking period. Hair cortisol concentration was assessed in 25-day-old hair samples taken from the frontal region of the head, analysing black and white coloured hair separately. Concentrations of cortisol metabolites were determined in faeces collected twice a week during the same period of time. There was a high correlation between cortisol values in faeces and cortisol in white colour hair samples but such correlation was not significant with the black colour hair samples. The intra- and inter-assay coefficients of variation were 4.9% and 10.6%, respectively. The linearity showed R 2=0.98 and mean percentage error of -10.8 ± 1.55%. The extraction efficiency was 89.0 ± 23.52% and the parallelism test showed similar slopes. Cortisol detection in hair by using EIA seems to be a valid method to represent long-term circulating cortisol levels in dairy cattle.

Hair cortisol concentrations (HCCs) and hair progesterone concentrations (HPCs) allow monitoring long-term retrospective steroid levels. However, there are still gaps in the knowledge of the mechanisms of steroid deposition in hair and its potential application in dairy cattle research. This study aimed to evaluate the potential uses of hair steroid determinations by studying the interrelations between HCC, HPC, physiological data from cows, and their milk production and quality. Cortisol and progesterone concentrations were analyzed in hair from 101 milking Holstein Friesian cows in a commercial farm. Physiological data were obtained from the 60 d prior to hair collection. Moreover, productive data from the month when hair was collected and the previous one were also obtained as well as at 124 d after hair sampling. Significant but weak correlations were found between HCC and HPC (r = 0.25, P0.0001) and between HPC and age (r = 0.06, P = 0.0133). High HCC were associated with low milk yields from the 2 previous months to hair sampling (P = 0.0396) and during the whole lactation (P0.0001). High HCC were also related to high somatic cell count (P = 0.0241). No effect of HCC on fat or protein content was detected. No significant correlations were detected between hair steroid concentrations and pregnancy status, days of gestation, parturition category (primiparous vs multiparous), number of lactations or days in milk. The relationship between physiological variables and HCC or HPC could depend on the duration of the time period over which hair accumulates hormones. Steroid concentrations in hair present high variability between individuals but are a potential tool for dairy cattle welfare and production research by providing a useful and practical tool for long-term steroid monitoring.

Oocyte cryopreservation by vitrification affects spindle integrity and causes oxidative stress induced by reactive oxygen species (ROS). Glutathione (GSH) ensures the correct assembly of microtubules, prevents ROS from attacking tubulin assembly, and ensures normal spindle function during meiosis. We hypothesised that addition of GSH ethyl ester (GSH-OEt), a cell-permeable GSH donor, to IVM medium before vitrification may improve oocyte cryotolerance by maintaining spindle morphology and preventing ROS production. For the IVM, viable cumulus-oocyte complexes were randomly distributed into two groups: 1) control: conventional IVM media with tissue culture medium-199 + 20% fetal calf serum + epidermal growth factor and 2) GSH-OEt: IVM medium supplemented with 5 mM GSH-OEt at 38.5°C in a 5% CO2 humidified air atmosphere. After 22 h of IVM, half of the oocytes from each group were vitrified/warmed using the Cryotop method (Kitazato Corp.). After 24 h of IVM, oocytes were denuded, fixed, and microtubule and chromosome distribution were analysed by immunofluorescence. To measure ROS, oocytes were denuded and incubated in 5 μM 2′,7′-dichlorodihydrofluorescein diacetate. Fluorescence was acquired, and resulting images were quantified with ImageJ software. A linear mixed effect followed by a pairwise comparison test (Tukey adjustment) were performed to analyse differences in meiotic spindle configuration and ROS production (P

For the successful application of vitrification technology to field conditions, the procedures for the warming and transfer of the cryopreserved bovine embryos should be as simple as possible. The device VitTrans, designed by our group, enables warming/dilution of embryos and their transfer directly to recipient females in field conditions (Morato and Mogas 2014 Cryobiology 68, 288). VitTrans vitrification protocol consists of an incubation in equilibration solution during 12 min followed by an exposure of 40 s to vitrification solution. However, there are other reports using similar vitrification devices where equilibration length is shorter than ours. This study aimed to improve VitTrans methodology by comparing two vitrification protocols: short equilibration (SE) and long equilibration (LE). A total of 63 in vitro-produced Day 7 blastocysts (IETS stage code 7) were randomly placed in an equilibration solution with 7.5% ethylene glycol + 7.5% dimethyl sulfoxide in holding medium (tissue culture medium-199 HEPES + 20% fetal calf serum) for either 3 min (SE) or 12 min (LE). Then, blastocysts were transferred to vitrification solution (15% ethylene glycol + 15% dimethyl sulfoxide + 0.5 M sucrose in holding medium) for 40 s, loaded onto the VitTrans device, plunged into liquid nitrogen, and covered with a 0.5 mL straw. Fresh nonvitrified blastocysts (n = 30) were set as control. For warming, the VitTrans was quickly submerged into a water bath at 45°C, while a syringe containing 0.3 mL of diluting solution (0.5 M sucrose in holding medium) at 45°C was injected through the hollow of the device. Blastocysts were then transferred to synthetic oviductal fluid medium and cultured for 24 h at 38.5% in a 5% CO2 and 5% O2 environment in a humidified atmosphere. Re-expansion rates were recorded 3 and 24 h after warming. Blastocysts were fixed and stained with SOX2 (Invitrogen) for inner cell mass (ICM) count, TUNEL (Roche) for apoptosis index assessment, and DAPI (Vector Laboratories) for total cell count (TCC). Images were captured using a Leica TCS SP5 confocal microscope (Leica Microsystems) and examined with Imaris 9.2 software (Oxford Instruments). Blastocyst survival rates were compared between groups using chi-squared test. Blastocyst TCC, ICM count, and apoptosis indices were analysed using analysis of variance. Significance was set at P ≤ 0.05. No differences were observed in re-expansion rate at 3 h postwarming (61.3 and 59.4% for SE and LE, respectively). However, significantly higher re-expansion rates were found after 24 h of culture for the blastocysts of the SE group (74.2%) when compared with the blastocysts of the LE group (65.7%). Blastocysts vitrified using the LE protocol produced the lowest TCC (115 ± 5.9; P ≤ 0.05), whereas TCC of the SE (152 ± 9.7) and fresh control (138 ± 8.6) treatments were similar. No differences were found in ICM count among groups. Nevertheless, apoptosis index was higher (P ≤ 0.05) in both vitrification groups when compared with fresh control. These results indicate that short equilibration vitrification not only improves VitTrans outcomes but adds efficiency by taking less time to perform. Supported by MCIU, Spain (Project AGL2016-79802-P and Grant BES-2017-081962).

The zona pellucida (ZP) is an extracellular envelope that surrounds mammalian oocytes. This coat participates in the interaction between gametes, induction of the acrosome reaction, block of polyspermy and protection of the oviductal embryo. Previous studies suggested that carnivore ZP was formed by three glycoproteins (ZP2, ZP3 and ZP4), with ZP1 being a pseudogene. However, a recent study in the cat found that all four proteins were expressed. In the present study, in silico and molecular analyses were performed in several carnivores to clarify the ZP composition in this order of mammals. The in silico analysis demonstrated the presence of the ZP1 gene in five carnivores: cheetah, panda, polar bear, tiger and walrus, whereas in the Antarctic fur seal and the Weddell seal there was evidence of pseudogenisation. Molecular analysis showed the presence of four ZP transcripts in ferret ovaries (ZP1, ZP2, ZP3 and ZP4) and three in fox ovaries (ZP2, ZP3 and ZP4). Analysis of the fox ZP1 gene showed the presence of a stop codon. The results strongly suggest that all four ZP genes are expressed in most carnivores, whereas ZP1 pseudogenisation seems to have independently affected three families (Canidae, Otariidae and Phocidae) of the carnivore tree.

High temperatures have negative effects on sperm quality leading to temporary or permanent sterility. The study tried to confirm the harmful effects of high temperatures on epididymal sperm cells in comparison with other temperatures (scrotal, environmental, and refrigeration temperatures), the main objective was the assessment of the addition of retinol as an antioxidant agent to improve sperm quality parameters. Testes from 10 bulls were collected from a slaughterhouse. Sperm cells were flushed from the cauda epididymis and deferent duct and assessed for sperm quality parameters at recovery. Afterward, sperm cell samples were exposed to one of four different temperatures (4 °C, 22 °C, 32 °C, and 41.5 °C for 3 hours) in presence or absence of retinol in the storage extender. Percentages of viability and morphologic abnormalities were determined using eosin-nigrosin staining. Acrosome integrity and sperm plasma membrane integrity were assessed by fluorescence Pisum sativum agglutinin lectin (FITC-PSA) staining and the hypo-osmotic swelling test, respectively. Total and progressive motility were analyzed by computer-assisted sperm analysis. Sperm quality parameters were mainly affected by high temperatures (41.5 °C). The addition of all-trans-retinol to the storage extender did not show any effect on sperm quality parameters. However, the percentage of sperm cells with altered acrosome was significantly reduced when retinol was present in the extender under heat stress conditions (41.5 °C). In conclusion, retinol might stabilize sperm acrosomal membrane in situations of oxidative stress because of high temperatures.

Heat stress is especially harmful for bovine ovarian follicle development and oocyte competence. In this study, we assessed the effects of heat shock on oocyte maturation in oocytes collected during the cold (February–March; n = 114) or warm (May–June; n = 116) periods of the year. In both cases, cumulus-oocyte complexes were matured under control (38 °C) and heat shock conditions (41.5 °C, 18–21 h of maturation). For each oocyte, nuclear stage, cortical granule distribution and steroidogenic activity of cumulus cells were evaluated. Based on the odds ratio, heat-shocked oocytes were 26.83 times more likely to show an anomalous metaphase II morphology. When matured under heat shock conditions, oocytes obtained in both seasons were similarly affected in terms of nuclear maturation, whereas a seasonal effect was observed on cytoplasmic maturation. For oocytes collected during the cold season, the likelihood to show an anomalous maturation was 25.96 times higher when exposed to the heat treatment than when matured under control conditions. By contrast, oocytes collected during the warm season matured under control or heat shock did not show significant risk of showing an anomalous cytoplasmic maturation. Our findings indicate an increased rate of premature oocytes in response to heat shock as well as a higher tolerance to this stress of oocytes harvested in the warm season compared with those collected in the colder period.

Animals under environmental thermal stress conditions have reduced fertility due to impairment of some mechanisms involved in their reproductive performance that are different in males and females. As a consequence, the most sensitive periods of time and the magnitude of effect of temperature on fertility can differ between sexes. The objective of this study was to estimate separately the effect of temperature in different periods around the insemination time on male and on female fertility by using the product threshold model. This model assumes that an observed reproduction outcome is the result of the product of 2 unobserved variables corresponding to the unobserved fertilities of the 2 individuals involved in the mating. A total of 7,625 AI records from rabbits belonging to a line selected for growth rate and indoor daily temperature records were used. The average maximum daily temperature and the proportion of days in which the maximum temperature was greater than 25°C were used as temperature descriptors. These descriptors were calculated for several periods around the day of AI. In the case of males, 4 periods of time covered different stages of the spermatogenesis, the transit through the epididymus of the sperm, and the day of AI. For females, 5 periods of time covered the phases of preovulatory follicular maturation including day of AI and ovulation, fertilization and peri-implantational stage of the embryos, embryonic and early fetal periods of gestation, and finally, late gestation until birth. The effect of the different temperature descriptors was estimated in the corresponding male and female liabilities in a set of threshold product models. The temperature of the day of AI seems to be the most relevant temperature descriptor affecting male fertility because greater temperature records on the day of AI caused a decrease in male fertility (-6% in male fertility rate with respect to thermoneutrality). Departures from the thermal zone in temperature descriptors covering several periods before AI until early gestation had a negative effect on female fertility, with the pre- and peri-implantational period of the embryos being especially sensitive (from -5 to -6% in female fertility rate with respect to thermoneutrality). The latest period of gestation was unaffected by the temperature. Overall, magnitude and persistency of the temperatures reached in the conditions of this study do not seem to be great enough to have a large effect on male and female rabbit fertility.

Embryo reduction may prevent the negative effects of twinning in dairy cattle; however, the technique may carry an additional risk of pregnancy loss. The aim of this study was to evaluate the effect on pregnancy maintenance of embryo reduction by manual amnion rupture in unilateral and bilateral twin pregnant cows. A secondary objective was to examine the dynamics of endocrine factors following the treatment. On Day 35-41 of gestation 55 cows bearing two live twin embryos (28 bilateral, 27 unilateral) were randomly assigned to a twin reduction group (n = 27; cows fitted with a progesterone releasing intra-vaginal device for 21 days after manual amnion rupture) or control group (n = 28; untreated cows). Pregnancy loss before Day 90 was recorded in nine control and eleven twin reduction cows (32.1% vs 40.7%, respectively, p = 0.508). Logistic regression models indicated that laterality was the only variable significantly affecting pregnancy loss. The pregnancy loss risk was 8.7 times higher for unilateral than for bilateral twin pregnancies (59.3% vs 14.3%, respectively, P0.001) yet was similar in the unilateral control and unilateral twin reduction cows (62.3% vs 53.8%, respectively, P = 0.581). In contrast, four of 14 cows with bilateral twin pregnancies undergoing twin reduction lost their pregnancies while no losses were recorded in control cows with bilateral pregnancies (P = 0.049). A rise in plasma progesterone concentration was detected on the day following treatment in the twin reduction group and concentrations remained high within the first week of treatment. Plasma pregnancy-associated glycoprotein-1 (PAG-1) concentrations fell between Day 35-41 and Day 42-48, regardless of treatment. Our findings indicate that embryo reduction by manual amnion rupture did not carry an additional risk of pregnancy loss for unilateral twin pregnancies, whereas it increased the risk of pregnancy failure in bilateral twin pregnancies. However, benefits of preventing cows from delivering twins might also be considered when assessing the success of embryo reduction in bilateral twin pregnancies.

Previous research indicated that the season during which oocytes are harvested affects quality of in vitro-produced embryos (Gupta et al. 2016 Anim. Reprod. Sci. 164, 162). In our own studies, sperm kinematics, membrane integrity, acrosome status, mitochondrial potential and reactive oxygen species were affected by season of semen collection (Sabés-Alsina et al. 2017 Vet. Rec. 180, 251). The aim of this study was to investigate effect of season of semen collection on in vitro fertilizing ability and embryo development of the same sperm samples from the sperm quality study, using lower than normal sperm doses to detect small differences between groups (Ward et al. 2003 Theriogenology 59, 1575). Frozen semen was available from 8 Holstein bulls kept outdoors in northern Spain, collected during winter, spring and summer. Bovine ovaries, Holstein and Swedish Red breeds, were obtained from an abattoir in spring. Oocytes were matured and fertilized in vitro with a low dose of frozen-thawed sperm (sperm:oocyte ratio 2500:1). After fertilization, presumptive embryos were evaluated at 44h for cleavage and on Day 8 for blastocyst development. Number of sperm binding to the zona pellucida and number of nuclei in developing blastocysts were assessed after staining with Hoechst 33342. There were 2 or 3 replicates per bull. For 555 oocytes inseminated, cleavage rates for winter, spring and summer semen collections were 42, 49 and 47%, respectively; blastocyst rates were 7, 12 and 8%; blastocyst cell numbers were 8.7, 10.2 and 7.8; and mean number of sperm bound was 0.70, 0.63 and 0.50. Although there were no differences (P>0.05) due to season of semen collection for any of these end points, individual bulls had considerable variation in cleavage rate. In conclusion, despite previously published differences in sperm quality with season from these bulls, ability of frozen-thawed sperm to fertilize bovine oocytes and initial embryo development in vitro were not affected by season of semen collection, at least for oocytes collected in spring. However, bull-to-bull variation in cleavage rate was high. The authors gratefully acknowledge the financial support of the Ministerio de Economía y Competitividad and FEDER (AGL2016-79802-P). M. Sabes-Alsina was supported by a PIF from the Universitat Autònoma de Barcelona and by the STS of the Epiconcept COST Action; J. M. Morrell was funded by the Swedish Research Council for the environment, agricultural sciences and spatial planning (FORMAS; 221-2010-1241) and the Swedish Farmers’ Association (SLF; 1330039).

The measure of corticosterone (CORT) in feathers has been recently recognized as a valid and easily obtainable measure of chronic glucocorti- coids secretion in avian species. This measure provides meaningful interpretations of how individuals respond to environmental perturbations. The growing interest of the public toward animal-food production welfare shows the need for improving and expanding objective tools to evaluate this issue. The present study evaluates whether it is possible to detect CORT in broiler feath- ers, and thus, assess if it would be a useful measure to study broiler welfare. Twenty-two broilers were ran- domly selected from an intensive farm. Four to 6 dorsal feathers were collected from each bird, and sex, weight, and morphological aspects of feather status were re- corded. We tested the feasibility for detecting CORT in broiler feathers by ELISA, which had never been done before, and an assay validation test was performed. No significant relationships were found between feather CORT concentrations and physiological variables such as sex, weight, and fault bars in broilers. To our knowl- edge, this is the first study that uses broiler feathers as a matrix that provides a retrospective record of their hypothalamic-pituitary-adrenal activity. Results indi- cate that ELISA is a valid tool to detect feather CORT levels in broilers.

Failures in fertilization or embryogenesis have been shown to be partly the result of poor semen quality. When AI is practiced, fertilization rate depends on the number and quality of spermatozoa in the insemination dose around the time of application. Individual variation in the male effect on fertility (success or failure to conceive; Fert) and prolificacy (total number of kids born per litter; TB) could also depend on these factors, and it could be better observed under limited conditions of AI, such as decreased sperm concentration, small or null preselection of ejaculates for any semen quality trait, or a long storage period of the AI doses. The aim of this research was to determine if an interaction existed between male genotype and the AI conditions for male effects on Fert and TB after AI was performed under different conditions. Fertility and TB were assumed to be different traits and were analyzed in 2 sets of independent analyses. In the first step, the different conditions were determined uniquely by the sperm dosage. Artificial insemination was performed at 10 and 40 × 10 6 spermatozoa/mL. In the second step, the different conditions were determined by all the factors involved in the AI process as a whole (conditions and duration of the storage period of the dose, genetic type of the female, and environmental conditions on the farm). Data from AI from the former experiment were analyzed with data from AI performed under different conditions. Threshold and linear 2-trait models were assumed for Fert and TB, respectively. The sperm dosage had a clear effect on Fert and TB, which favored the greater dosage (+0.13% and +1.25 kids born, respectively). Prolificacy was more sensitive to sperm reduction than was fertility. Male heritabilities for Fert were 0.09 for both sperm dosages, and were 0.08 and 0.06 for male TB with a smaller and larger sperm dosage, respectively. No genotype x sperm dosage interaction was found. Therefore, the same response to selection to improve male Fert and TB could be achieved at any sperm concentration. However, an interaction between male genotype and the AI conditions as a whole seemed to exist, indicating that the AI conditions for selection for Fert and TB could be modified to maximize genetic progress. Consequently, the optimization of a breeding program for male Fert and TB under a given set of semen utilization conditions is achievable.

This study analyses reproductive parameters resulting from 74 parturitions in a population of captive collared peccaries in the eastern Amazon (Belém, State of Pará, Brazil) during a 65-month survey. Parturitions were homogeneously distributed throughout the year. The average litter size was 1.85 newborns per parturition. Collared peccary females had a newborn sex ratio of 52.6% females and 47.4% males. The mean age at first parturition was 639, although, the earliest first parturition occurred at age of 381 days. Estimated parturition-conception interval was 58 days. Mean farrowing interval was 196 days and mean production was 1.03 litters and 1.86 newborns per year per female. Mortality rate in newborns less than 2 months old was 26.9% of the total newborn population. Most identified causes of death were abandonment by the mother (22.2%) and trauma due to aggression (13.9%). Newborn deaths occurred mainly during the first two days of life. This study suggests that the collared peccary possesses interesting reproductive parameters for its introduction in captive breeding programmes in the Amazon region. © 2006 Published by Elsevier B.V.

The aim of the present study was to establish whether temperature-humidity index values, as a measure of heat comfort, from Days 1 to 40 of gestation could be associated with the pregnancy loss rate in high producing dairy cows. Data from 1391 pregnancies were recorded. Pregnancy was diagnosed by transrectal ultrasonography between Days 34 and 45, and again 90 days after insemination. Pregnancy loss was assumed when the second pregnancy diagnosis on Day 90 proved negative and was registered in 7.8% (108/1391) of pregnancies. Mean and maximum temperature-humidity index values were established for each cow for Days 0 (day of insemination), 1, 2 and 3 after insemination, and averages established for Days 0-3, 0-10, 11-20, 21-30 and 31-40 after insemination. Cow and management variables previously found to be significantly correlated with the early fetal loss in the same geographical area were also recorded. The relative contribution of each factor to the probability of pregnancy loss was determined using logistic regression models. Based on the odds ratio, a strong association with pregnancy loss of the factors warm period of pregnancy (warm period-May to September versus cool-October to April), twin pregnancy (as negative factors: odds ratios 3.1 and 3.4, respectively) and an additional corpus luteum (as a positive factor: odds ratio 0.32) was confirmed. The likelihood of pregnancy loss increased by a factor of 1.05 for each additional unit of the mean maximum temperature-humidity index from Days 21 to 30 of gestation. Logistic regression analysis revealed no significant effects of temperature-humidity index values for the remaining gestation periods. Our results indicate that heat stress can compromise the success of gestation during the peri-implantation period, such that high temperature-humidity index values for the period 21-30 days of gestation are a risk factor for subsequent early fetal loss.

The effect of intravenous cloprostenol treatment at the time of insemination on reproductive performance was consecutively evaluated in three different subpopulations of high producing lactating dairy cows: Study (1) early postpartum synchronized and fixed-time inseminated (about 50 days in milk) cows (n = 379: 187 control and 192 treated cows); Study (2) presumed high fertility cows first inseminated between 90 and 120 days postpartum (n = 248: 124 control and 124 treated cows); and Study (3) heat stressed repeat breeder cows (n = 183: 93 control and 90 treated cows). Data were analyzed using multiple regression methods. Study 1: Parity (primiparous versus multiparous), milk production, body condition score at AI, insemination season (cool versus warm period) and treatment were included in the analysis as potential factors affecting ovulation, double ovulation, return to estrus, and pregnancy to first AI and to second AI (first AI plus return AI) rates. Logistic regression analysis indicated that the final model for ovulation rate only included the interaction (P = 0.002) between insemination season and treatment. Cloprostenol treatment at insemination led to a 4.2-fold increase in the ovulation rate in cows inseminated during the warm period. There were no significant effects of treatment, parity, milk production, body score or the insemination season on the return to estrus rate. The only variables included in the final logistic model for double ovulation and pregnancy to first AI rates were treatment and season, respectively. Treatment led to a 2.6-fold increase (P = 0.001) in the double ovulation rate, whereas cows inseminated in the warm period were 2.1 times less likely (P = 0.007) to become pregnant at first AI compared to those inseminated in the cool season. The variables included in the final logistic model for the pregnancy rate to second AI were treatment and season. Cloprostenol given at AI increased the risk of pregnancy 1.9 times (P = 0.002), and cows inseminated during the warm season were two times less likely to become pregnant (P = 0.003). No significant interactions were found among these three dependent variables (double ovulation and pregnancy to first and to second AI rates). Study 2: Logistic regression analysis of all the dependent variables: return to estrus, and pregnancy to first and to second AI (first AI plus return to AI) rates indicated no significant effects of treatment, parity, days in milk, milk production or body score at AI. No significant interactions were found. Study 3: The final model for the pregnancy rate only included the interaction between parity (primiparous versus multiparous) and treatment. Days in milk, milk production and insemination number showed no significant effect on pregnancy rate. Cloprostenol treatment at insemination increased the pregnancy rate in primiparous repeat breeder cows (odds ratio: 3.6). The treatment group and parity showed significant (P < 0.0001) interaction. This interaction suggests that cloprostenol treatment of primiparous cows at insemination might enhance pregnancy yet have no effect in multiparous cows. Our findings indicate that cloprostenol administered at insemination promotes ovulation and double ovulation in lactating dairy cows. Cloprostenol treatment showed no benefit in cows with acceptable reproductive performance, suggesting that cloprostenol treatment at AI may only be useful in cows in which stress factors affect ovulation and in repeat breeder cows.

Previous reports have demonstrated the beneficial effect of antifreeze glycoprotein supplementation during oocyte vitrification on preventing ice crystal formation and thus enhancing developmental competence after vitrification-warming. Pseudomonas sp. ID1, a bacterium isolated from marine sediment from Antarctica, produces an exopolysaccharide, M1 EPS, as a cold adaptation mechanism. Despite numerous studies on structural and morphological damages induced by cryopreservation in oocytes, few studies have focused on the impact of vitrification on the expression pattern of genes during early embryo development. In the present study, the expression patterns of 6 genes (BAX, BCL2-like 1, DNMT3A, UBE2A, SCLC2A3, and HDAC1) were investigated in Day 8 blastocysts resulting from in vitro-matured oocytes vitrified/warmed in media supplemented with various concentrations of M1 EPS. After 21 h of IVM, 1,062 oocytes were vitrified/warmed in media supplemented with 0, 0.001, 0.01, 0.1, and 1 mg mL−1 M1 EPS. At 24 h of IVM, oocytes were in vitro fertilized and in vitro cultured and the resulting blastocysts were harvested at Day 8 for RNA extraction and qPCR analysis. Fresh, non-vitrified oocytes were used as a control. Analysis of gene expression was performed through Kruskall-Wallis test and followed by Mann-Whitney test, and the level of significance was set at P ≤ 0.05. No significant differences were detected in relative mRNA abundance for SLC2A3, UBE2A, or HDAC-1 between blastocysts derived from vitrified oocytes, regardless of M1 EPS treatment. Expression of DNMT3A was significantly higher in embryos obtained from oocytes vitrified and warmed with 0.1 mg mL−1 M1 EPS compared with other treatment groups. However, no differences in DNMT3A expression were observed when the other vitrified groups were compared. The relative abundance of BAX transcript in embryos from oocytes vitrified in media supplemented with 0.1 mg mL−1 M1 EPS was higher than that in 0 or 0.001 mg mL−1 groups. Embryos from 0.01 and 0.1 mg mL−1 groups showed higher BCL2-like 1 mRNA abundance than those from the 0, 0.001, and 1 mg mL−1 groups. Whereas blastocysts from oocytes vitrified with 0.01 mg mL−1 M1 EPS exhibited the lowest BAX:BCL2-like 1 ratio, no significant differences in BAX:BCL2-like 1 ratio were observed between the other treatments. The significantly lower BAX:BCL2 ratio observed in blastocysts obtained from oocytes vitrified with 0.01 mg mL−1 M1 EPS could be indicative for a better embryo quality. Although optimizing the use of M1 EPS may benefit oocyte cryopreservation protocols, further research is required to clarify the exact mechanism through which it exerts its protective role. This study was supported by the Spanish Ministry of Science and Innovation (Project AGL2016-79802-P and grant CTQ2014-59632-R).

International audience; High temperatures have negative effects on sperm quality leading to temporary or permanent sterility. The aim of the study was to assess the effect of long exposure to summer circadian heat stress cycles on sperm parameters and the motile subpopulation structure of epididymal sperm cells from rabbit bucks. Twelve White New Zealand rabbit bucks were exposed to a daily constant temperature of the thermoneutral zone (from 18 °C to 22 °C; control group) or exposed to a summer circadian heat stress cycles (30 °C, 3 h/day; heat stress group). Spermatozoa were flushed from the epididymis and assessed for sperm quality parameters at recovery. Sperm total motility and progressivity were negatively affected by high temperatures (P < 0.05), as were also specific motility parameters (curvilinear velocity, linear velocity, mean velocity, straightness coefficient, linearity coefficient, wobble coefficient, and frequency of head displacement; P < 0.05, but not the mean amplitude of lateral head displacement). Heat stress significantly increased the percentage of less-motile sperm subpopulations, although the percentage of the high-motile subpopulation was maintained, which is consistent with the fact that no effect was detected on fertility rates. However, prolificacy was reduced in females submitted to heat stress when inseminated by control bucks. In conclusion, our results suggest that environmental high temperatures are linked to changes in the proportion of motile sperm subpopulations of the epididymis, although fertility is still preserved despite the detrimental effects of heat stress. On the other hand, prolificacy seems to be affected by the negative effects of high temperatures, especially by altering female reproduction.

International audience; Heat stress (HS) in mammals is a determining factor in the deterioration of spermatogenesis and can cause infertility. The aim of this study was to evaluate the effect of continuous summer circadian cycles on semen production, sperm cell features, fertility, prolificacy, and fecal cortisol metabolites from rabbits kept under an in vivo HS model. We split randomly 60 New Zealand White rabbits into two temperature-controlled rooms: The control group was maintained at comfort temperature (18 degrees C-22 degrees C) and an HS group, where the environmental temperature was programmed to increase from 22 degrees C to 31 degrees C and be maintained for 3 hours to this temperature at the central part of the day. Fecal cortisol metabolites were assessed to evaluate the stress conditions. Seminal parameters were analyzed. Although animals exposed to HS showed higher values of fecal cortisol metabolites (P = 0.0003), no differences were detected in fertility or prolificacy. Semen samples from HS males showed a significant decrease (P < 0.05) with respect to the controls in the percentage of viable spermatozoa (80.71% vs. 74.21%), and a significant (P < 0.01) increase in the percentage of acrosomic abnormalities (22.57% vs. 36.96%) and tailless spermatozoa (7.91% vs. 12.83). Among motility parameters, no differences were found. This study describes a model of HS simulating a continuous summer daily cycle that allows periods of time to recover as it occurs under natural conditions. Although negative effects have been detected in several sperm parameters, fertility and prolificacy were not affected, suggesting a recovery of the reproductive function when normal conditions are reestablished.

This review focuses on factors associated with the development of intraperitoneal insemination in mammals. Findings to date indicate that fertility improves as the sperm cell concentration rises, but that the optimal sperm number differs in each species. Sperm washing before intraperitoneal insemination favours fertility. Peritoneal fluid shows a variable effect on spermatozoa, depending on the hormonal status of the female. The optimal time for insemination appears to be just prior to ovulation. The technique may be performed either through the abdominal or the vaginal wall. Verification of sperm deposition in the proximity of the ovaries improves fertility rates. Although associated with some risk of infection and an immune reaction against spermatozoa, the intraperitoneal technique rarely gives rise to severe anaphylactic shock, peritonitis, adhesion formation and the production of anti-sperm antibodies and these complications may be prevented by adequate sperm pretreatment and antibiotic therapy. The success of intraperitoneal insemination in humans, with results comparable with those of intrauterine insemination in the treatment of infertility, suggest the potential use of this technique in domestic mammals, especially in those in which intrauterine insemination poses practical difficulties. Some of the methods applied in human intraperitoneal insemination, such as confirming the position of the needle in the peritoneal cavity, and sperm pre-treatments might also improve results in domestic species. Conversely, the use of the animal model should help to develop some aspects of this technique in humans.

The brush-tailed porcupine Atherurus africanus is an important source of meat for local people in tropical forested areas of Central Africa. Information on the biology of this species in free-ranging conditions is scarce. Data on its reproduction and productivity are essential to establish accurate management practices in the exploitation of this rodent. In order to provide data on the reproductive physiology and to assess the prolificacy of the brush-tailed porcupine in the wild, the structural and functional anatomy of the ovaries of free-ranging females were studied. Twenty-two female carcasses were obtained from various markets in Libreville, Gabon, between August 1995 and February 1997. Reproductive tracts were macroscopically examined for the presence of embryos or foetuses. The ovaries were embedded in paraffin wax or plastic and examined by light microscopy. The morphology and constituent cell types of the ovaries were similar to those described for other hystricognath rodents. All pregnant females had only one embryo or foetus and the number of corpora lutea (CL) per female was 19.9 a 5.4 (mean diameter a sd, 1709 a 421 mm), including one CL of pregnancy (diameter, 4821 a 1364 mm). Also, 8.9 a 6.8 accessory CL per female in the pre-implantation and embryonic stages of pregnancy (diameter, 1629 a 463 mm) were observed. The average active luteal volume was high (115.6 a 73.5 mm 3 ). The mean number of antral follicles in the adult females was 24.6 a 9.9. The study confirms that the brush-tailed porcupine is a mono-embryonic species, as observed in captive breeding experiences, but polyovular, suggesting important ovum mortality. This unusual reproductive feature in a mammalian species gives this rodent a low reproductive productivity. Therefore, A. africanus could be more susceptible to the effects of hunting than generally thought.

Anestrus is common during the postpartum period in high-producing dairy cows. In a previous investigation, we were able to diagnose persistent follicles of 8 to 12 mm in anestrous cows. This report describes 2 consecutive studies. The objectives of the first were to 1) assess the association of persistent follicles with anestrus; and 2) evaluate 2 therapeutic treatments. In the second study, we compared the effectiveness of the best treatment established in Study 1 with the Ovsynch protocol. For Study 1, anestrous cows were considered to have a persistent follicle if it was possible to observe a single follicular structure8 mm in the absence of a corpus luteum or a cyst in 2 ultrasonographic examinations performed at an interval of 7 d. At diagnosis (Day 0), cows were assigned to 1 of 3 treatment groups. Cows in Group GnRH/PGF (n=17) were treated with 100 microg GnRH i.m., and 25 mg PGF2alpha i.m. on Day 14. Cows in Group PRID (n=18) were fitted with a progesterone releasing intravaginal device (PRID, containing 1.55 g of progesterone) for 9 d and were given 100 microg GnRH i.m. at the time of PRID insertion, and 25 mg PGF2alpha i.m. on Day 7. Cows in Group Control (n=18) received no treatment. The animals were inseminated at observed estrus and were monitored weekly by ultrasonography until AI or 5 weeks from diagnosis. Blood samples were also collected on a weekly basis for progesterone determination. The mean size of persistent follicles on Day 0 was 9.4 +/- 0.04 mm. Progesterone levels were0.2 ng/mL during the first 35 d in 16 of 18 Control cows. Cows in the PRID group showed a lower persistent follicle rate (16.7%70.6%88.9%; P0.0001; PRID vs GnRH/PGF vs Control, respectively); a higher estrus detection rate (83.3%29.4%11.1%; P0.0001) and a higher pregnancy rate (27.8%5.9%0%; P = 0.02). For the second study, 145 cows with persistent follicles were randomly assigned to 1 of 2 treatment groups: cows in Group Ovsynch (n=73) were treated with 100 microg GnRH i.m. on Day 0, 25 mg PGF2alpha i.m. on Day 7, and 100 microm GnRH i.m. 32 h later. Cows in this group were inseminated 16 to 20 h after the second GnRH dose (Ovsynch protocol). Cows in Group PRID (n=72) were treated as those in the PRID group of Study 1, and were inseminated 56 h after PRID removal. Cows in the PRID group showed a higher ovulation rate (84.8%8.2%: P0.0001); a higher pregnancy rate (34.2%4.1%; P0.0001) and lower follicular persistence rate (22.2%63%; P0.0001) than those in Ovsynch. Our results indicate that persistent follicles affect cyclic ovarian function in lactating dairy cows. Cows with persistent follicles can be successfully synchronized and time inseminated using progesterone, GnRH and PGF2alpha but show a limited response to treatment with GnRH plus PGF2alpha.

The present study was conducted to evaluate rheological properties of the vaginal fluid in dairy cows. Thixotropy and flow behavior were evaluated. Vaginal fluid samples were collected in 8 cows at the beginning of a natural estrus and then every 8 h for 24 h. Vaginal samples were also collected from 208 cows at the time of insemination within 12 to 24 h interval after the onset of a natural estrus. Time-dependent variations were registered during the estrus. The limit shear stress for flow production (To), the shear stress of equilibrium (Te) and the consistency index (K) decreased at 8 and 16 h and markedly increased 24 h after the beginning of estrus. At the time of insemination, structural variation was registered and the fluids were considered to be thixotropic in samples of 181 cows (87%); in the remaining samples (27 cows), structural variation was not registered and the fluids were considered to be nonthixotropic. For flow behavior values, 178 samples (85.6%) presented non-Newtonian behavior, while 30 behaved as Newtonian fluids. The mean values of the TO and the Te were 6.1 ± 0.69 and 1.9 ± 0.69 pascals, respectively. Consistency index (K) mean was 364.9 ± 41.4 milipascals per secondn Our data show an increase of fluidity at the middle of the estrus period in the cow and a wide variation of rheological measurements of vaginal fluid at the time of insemination.

[EN] Contents In this study, lectin histochemistry was performed on paraffin sections to compare carbohydrate expression of oviductal isthmus and uterine endometrium in rabbits during early embryo development. Rabbit embryos are surrounded not only by the zona pellucida but also by tubal secretion-derived mucinous coat material, the mucin coat. Twenty sexually mature females were euthanized at 0 (pre-ovulatory group) and 24, 72 and 96h after insemination (pseudopregnancy group). The following lectin-binding agents were used: Arachis hypogaea, Peanut (PNA) to label galactosyl (-1,3)N- acetyl-galactosamine, Dolichos biflorus Agglutinin (DBA) to label galactosyl (-1,4)N- acetyl-galactosamine, Lens curinaris (LCA) to label --mannose, -d-glucose and Pisum sativum agglutinin (PSA) to label -d-mannose, -d-glucose. Blood was collected by cardiac puncture, and direct enzyme immunoassay technique was used to measure progesterone concentration. A significant increase in total plasma progesterone concentrations was detected at 96h post-ovulation when compared with 0, 24 and 72h post-ovulation (2.9 +/- 0.5 vs 0.5 +/- 0.15, 1.6 +/- 0.5 and 1.5 +/- 0.4ng/ml, at 96h vs 0, 24 and 72h post-ovulation, respectively). No differences between pre-ovulatory and pseudopregnant females were observed for glycoprotein localization in isthmus. In contrast, in the endometrium, differences in the glycoprotein detection between pre-ovulatory and pseudopregnant stages were detected. PNA to label galactosyl (-1,3)N- acetyl-galactosamine was not detected at the pre-ovulatory stage, but its presence was detected at 24h after ovulation. Both PSA and LCA to label -d-mannose, -d-glucose were only detected at 72h after ovulation. DBA detection was similar for all stages of the reproductive cycle. Therefore, N-acetyl-galactosamine secreted from isthmus could be involved in the formation of the embryonic mucin coat. d-galactose (PNA), d-glucose and d-mannose (PSA and LCA) might be crucial for the implantation period., Francisco Marco Jimernez was supported by Polytechnic University of Valencia research and development programme (3141/2008). English text version was revised by N. Macowan English Language Service.

This research aimed at assessing the efficacy of non-parametric procedures to improve the classification of the ejaculates in the artificial insemination (AI) centers according to their fertility rank predicted from characteristics of the AI doses. A total of 753 ejaculates from 193 bucks were evaluated at three different times from 5 to 9 months of age for 21 seminal variables (related to ejaculate pH and volume, sperm concentration, viability, morphology and acrosome reaction traits, and dose characteristic) and their corresponding fertility score after AI over crossbred females. Fertility rate was categorized into five classes of equal length. Linear Regression (LR), Ordinal Logistic Regression (OLR), Support Vector Regression (SVR), Support Vector Ordinal Regression (SVOR), and Non-deterministic Ordinal Regression (NDOR) were compared in terms of their predictive ability with two base line algorithms: MEAN and MODE which always predict the mean and mode value of the classes observed in the data set, respectively. Predicting ability was measured in terms of rate of erroneous classifications, linear loss (average of the distance between the predicted and the observed classes), the number of predicted classes and the F1 statistic (which allows comparing procedures taking into account that they can predict different number of classes). The seminal traits with a bigger influence on fertility were established using stepwise regression and a nondeterministic classifier. MEAN, LR and SVR produced a higher percentage of wrong classified cases than MODE (taken as reference for this statistic), whereas it was 6%, 13% and 39% smaller for SVOR, OLR and NDOR, respectively. However, NDOR predicted an average of 2.04 classes instead of one class predicted by the other procedures. All the procedures except MODE showed a similar smaller linear loss than the reference one (MEAN) SVOR being the one with the best performance. The NDOR showed the highest value of the F1 statistic. Values of linear loss and F1 statistics were far from their best value indicating that possibly, the variation in fertility explained by this group of semen characteristics is very low. From the total amount of traits included in the full model, 11, 16, 15, 18 and 3 features were kept after performing variable selection with the LR, OLR, SVR, SVOR and NDOR methods, respectively. For all methods, the reduced models showed almost an irrelevant decrease in their predictive abilities compared to the corresponding values obtained with the full models, This research was supported by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA, Madrid, Spain) project RTA2005-00088-CO2

Ovarian stimulation with equine chorionic gonadotropin (eCG) is largely used in animal reproductive technologies to provide a larger number of oocytes and embryos and to improve the reproductive outcome. However, the consequences of maternal treatment with eCG on embryo gene expression patterns are not widely studied. The aim of this work was to assess the ovarian response (preovulatory follicular population, oocyte maturation, ovulation rate, and serum steroid concentrations), the early embryo survival and gene expression patterns of a panel of quality-genes involved in glucose intake, oxidative stress, apoptosis, proliferation, implantation, and fetal growth in embryos of lactating rabbits treated with eCG. A total of 34 primiparous rabbit does (Oryctolagus cuniculus) were randomly distributed at Day 23 postpartum into a treatment group receiving a unique nonsuperovulatory dose (25 IU) of eCG (eCG group; N = 17 does); or a control group without eCG treatment previously to artificial insemination (control group; N = 17 does). After 48 hours, 8 does of each group were euthanized and their ovarian response was studied. The rest of animals were artificially inseminated and their ovulation was induced with a GnRH analogue. Embryos were recovered 3.5 days later. The oocytes retrieved for in vitro maturation showed no differences in metaphase II rate in both experimental groups, although oocyte cytoplasmic maturation, in terms of cortical granule migration rate, was improved in eCG-treated does (P < 0.05). The mean number of preovulatory follicles was similar between groups but the ovulation rate was significantly higher in eCG-treated does compared with does not stimulated (P < 0.05). No differences were found in serum estradiol and progesterone concentrations of does the day of oocyte and embryo recovery, respectively. However, progesteroneestradiol ratio was slightly increased in eCG group on embryo recovery day (P = 0.1). The percentage of embryos recovered at the blastocyst stage was also increased in eCG-treated does (P < 0.05), nevertheless, there were no differences in the gene expression patterns of candidate genes SLC2A4, IGF1R, IGF2R, SHC1-SHC, TP53, PTGS2, and PLAC8; except for the transcripts of SOD1 mRNA which were downregulated in eCG-derived embryos (P < 0.05). In conclusion, the administration of eCG improves ovulation rate, oocyte cytoplasmic maturation, and blastocyst formation in primiparous rabbit does inseminated on Day 25 postpartum. Although it seems not to influence the gene expression patterns studied, a lower antioxidant defense of embryos developed after the maternal eCG treatment is suggested. © 2013 Elsevier Inc.